The present invention relates to a thermostable enzyme which is a DNA polymerase obtainable from Anaerocellum thermophilum. 
Heat stable DNA polymerases (EC 2.7.7.7. DNA nucleotidyltransferase, DNA-directed) have been isolated from numerous thermophilic organisms (for example: Kaledin et al., 1980, Biokimiya Vol. 45, p. 644-651; Kaledin et al., 1981, Biokimiya Vol. 46, p. 1247-1254; Kaledin et al.,1982, Biokimiya Vol. 47, p. 1515-1521; Ruttimann, et al., 1985, Eur. J. Biochem. Vol. 149, p. 41-46; Neuner et al., 1990, Arch. Microbiol. Vol. 153, p. 205-207.)
For some organisms, the polymerase gene has been cloned and expressed (Lawyer et al., 1989, J. Biol. Chem. Vol. 264, p. 6427-6437; Engelke et al., 1990, Anal. Biochem. Vol. 191, p. 396-400; Lundberg et al., 1991, Gene, Vol. 108, p. 1-6; Kaledin et al., 1980 Biokimiya Vol. 44, p. 644-651; Kaledin et al., 1981, Biokimiya Vol. 46, p. 1247-1254; Kaledin et al., 1982, Biokimiya Vol. 47, p. 1515-1521; Ruttimann, et al., 1985, Eur. J. Biochem. Vol. 149, p. 41-46; Neuner et al., 1990, Arch. Microbiol. Vol. 153, p. 205-207; Perler et al., 1992, Proc. Natl. Acad. Sci. USA Vol. 89, p. 5577).
Thermophilic DNA polymerases are increasingly becoming important tools for use in molecular biology and there is growing interest in finding new polymerases which have more suitable properties and activities for use in diagnostic detection of RNA and DNA, gene cloning and DNA sequencing. At present, the thermophilic DNA polymerases mostly used for these purposes are from Thermus species like Taq polymerase from T. aquaticus (Brock et al 1969, J. Bacteriol. Vol. 98, p. 289-297).
Reverse transcription is commonly performed with viral reverse transcriptases like the enzymes isolated from Avian myeloblastosis virus or Moloney murine leukemia virus, which are active in the presence of Magnesium ions but have the disadvantages to possess RNase H-activity, which destroys the template RNA during the reverse transcription reaction and have a temperature optimum at 42xc2x0 C. or 37xc2x0 C., respectively.
Alternative methods are described using the reverse transcriptase activity of DNA polymerases of thermophilic organisms which are active at higher temperatures. Reverse transcription at higher temperatures is of advantage to overcome secondary structures of the RNA template which could result in premature termination of products. Thermostable DNA polymerases with reverse transcriptase activities are commonly isolated from Thermus species. These DNA polymerases however, show reverse transcriptase activity only in the presence of Manganese ions. These reaction conditions are suboptimal, because the presence of Manganese ions lowers the fidelity of the DNA polymerase transcribing the template RNA.
Therefore, it is desirable to develop a reverse transcriptase which acts at higher temperatures to overcome secondary structures of the template and is active in the presence of Magnesium ions in order to prepare cDNA from RNA templates with higher fidelity.
The present invention addresses these needs and provides a purified DNA polymerase enzyme (EC 2.7.7.7.) active at higher temperatures which has reverse transcriptase activity in the presence of magnesium ions. The invention comprises a DNA polymerase isolated from Anaerocellum thermophilum DSM 8995, deposited on the Deutsche Samnulung von Mikro-organismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. In a further aspect the invention comprises a DNA polymerase that catalyses the template directed polymerisation of DNA and posess 5xe2x80x2-3xe2x80x2-polymerase activity, 5xe2x80x2-3xe2x80x2-exonuclease activity and no substantial 3xe2x80x2-5xe2x80x2-exonuclease activity.
The polymerase according to the present invention retains at least 90% of its activity after incubation for 30 Minutes at 80xc2x0 C. in absence of stablilizing detergents.
In a further aspect the invention comprises a DNA polymerase having a molecular mass of about 96 to 100 kDa as determined by in situ activity PAGE analysis.
In a futther aspect the invention comprises a DNA a polymerase having reverse transcriptase activity in the presence of magnesiums ions and in the substantial absence of maganese ions. The polymerase according to the present invention exhibits a Mg2+ dependent reverse transcriptase activity of more than 30% relative to the DNA polymerase activity which is set to 100%. In further aspect the present invention comprises a thermostable DAN polymerase wherein said polymerase exhibits a reverse transcriptaqse activity which is Mn2+ dependent. The Mn2+ dependent reverse transcriptase activity is more than 60% relative to the DNA polymerase activity.
In further aspect the invention comprises a thermostable reverse transcriptase. The thermostable reverse transcriptase retains more than 80% after incubation for 60 minutes at 80xc2x0 C.
Moreover, DNA encoding the 96.000-100.000 daltons thermostable DNA polymerase obtainable from Anearocellum thermophilum has been isolated and which allows to obtain the thermostable enzyme of the present invention by expression in E. coli. the entire Anearocellum thermophilum DNA polymerase coding sequence is depicted below as SEQ ID NO. 7. The recombinant Anearocellum thermophilum DNA polymerase also possesses 5xe2x80x2-3xe2x80x2polymerase activity, no substantial 3xe2x80x2-5xe2x80x2-exonuclease activity, 5xe2x80x2-3xe2x80x2-exonuclease activity and a reverse transcriptase activity which is a Mg2+ dependent.
Anaerocullum thermophilum was isolated from a hot spring in the Valley of Geyser in Kamchatka (V. svetlichny et al. Mikrobilogiya, Vol. 59, No. 5 p. 871-879, 1990). Anaerocullum thermophilum was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig under the terms of the Budapest Treaty and received DSM Accession Number 8995. The thermostable polymerase isolated from Anaerocellum thermophilum has a molecular weight of 96 to 100 kDa and retains more than 90% of activity after heating to 80xc2x0 C. for 30 minutes in absence of stabilizing detergents. The thermostable enzyme possesses a 5xe2x80x2-3xe2x80x2 polymerase activity and a reverse transcriptase activity which is Mn++ as well as Mg++-dependent. The thermostable enzyme may be native or recombinant and may be used for first and second strand cDNA synthesis, in cDNA cloning, DNA sequencing, DNA labeling and DNA amplification.
The present invention provides improved methods for the replication and amplification of deoxyribonucleic (DNA) and ribonucleic acid (RNA) sequences. These improvements are achieved by the discovery and application of previously unknown properties of thermoactive DNA polymerases. In a preferred embodiment, the invention provides a method for synthesizing a complementary DNA copy from an RNA template with a thermoreactive DNA polymerase. In another aspect, the invention provides methods for amplifying a DNA segment from an RNA or DNA template using a thermostable DNA polymerase (RT-PCR or PCR).
The term xe2x80x9creverse transcriptasexe2x80x9d describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
For recovering the native protein Anaerocellum thermophilum may be grown using any suitable technique, such as the technique described by Svetlichny et al., 1991, System. Appl. Microbiol. Vol. 14, p. 205-208. After cell growth one preferred method for isolation and purification of the enzyme is accomplished using the multi-step process as follows:
The cells are thawed, suspended in buffer A (40 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 0.4 M NaCl, 10 mM Pefabloc(trademark) SC (4-(2-Aminoethyl)-benzolsulfonyl-fluorid, Hydrochlorid) and lysed by twofold passage through a Gaulin homogenizer. The raw extract is cleared by centrifugation, the supernatant dialyzed against buffer B (40 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 10% Glycerol) and applied onto a column filled with Heparin-Sepharose (Pharmacia). In each case the columns are equilibrated with the starting solvent and after application of the sample the columns are washed with the threefold of their volume with this solvent. Eluation of the first column is performed with a linear gradient of 0 to 0.5 M NaCl in Buffer B. The fractions showing polymerase activity are pooled and ammonium sulfate is added to a final concentration of 20%. This solution is applied to a hydrophobic column containing Butyl-TSK-Toyopearl (TosoHaas). This column is eluted with a falling gradient of 20 to 0% ammonium sulfate. The pool containing the activity is dialyzed and again transferred to a column of DEAE-Sepharose (Pharmacia) and eluted with a linear gradient of 0-0.5 M NaCl in buffer B. The fourth column contains Tris-Acryl-Blue (Biosepra) and is eluted as in the preceding case. Finally the active fractions are dialyzed against buffer C (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7.0 mM 2-mercaptoethanol, 100 mM NaCl, 50% Glycerol).
DNA polymerase activity was either measured by incorporation of 32P-dCTP or by incorporation of digoxigenin labeled dUTP into the synthesized DNA. Detection and quantification of the incorporated digoxigenin was performed essentially as described in Holtke, H.-J.; Sagner, G. Kessler, C. and Schmitz, G., 1992, Biotechniques Vol. 12, p. 104 -113.
Reverse transcriptase activity was measured using oligo dT primed poly A template by incorporation of either 32P-dTTP or digoxigenin-labeled dUTP into the complementary strand. Detection of the incorporated digoxigenin was performed in analogy to the procedure used for detection of DNA polymerase activity.
In situ PAGE analysis of polymerase activity and reverse transcriptase activity was performed essentially according to the method described by Spauos A. and Hxc3xcbscher U., 1983, Methods in Enzymology Vol. 91 p. 263-277. Some minor, but essential modifications to the original method are, that the renaturation of the SDS-denatured polypeptides is performed in the presence of magnesium ions (3 mM) and dATP (0.5-1 xcexcM) to assist refolding.
The thermostable enzyme of this invention may also be produced by recombinant DNA techniques, as the gene encoding this enzyme has been cloned from Anaerocellum thermophilum genomic DNA. In a firer aspect the invention includes a recombinant plasmid comprising the vector pASK75 carrying the Anaerocellum thermophilum DNA polymerase gene and designated pAR10.
The isolation of the recombinant clone expressing DNA polymerase from Anaerocellum thermophilum includes the following steps: chromosomal DNA from Anaerocellum thermophilum is isolated by treating the cells with detergent e.g. SDS and a proteinase e.g. Proteinase K. The solution is extracted with phenol and chloroform and the DNA purified by precipitation with ethanol. The DNA is dissolved in Tris/EDTA buffer and the gene encoding the DNA polymerase is specifically amplified by the PCR technique using two mixed oligonucleotides (primer 1 and 2). These oligonucleotides, described in SEQ ID NO.: 1 and SPQ ID NO.: 2, were designed on the basis of conserved regions of family A DNA polymerases as published by Braithwaite D. K. and Ito J., 1993, Nucl. Acids Res. Vol. 21, p. 787-802. The specifically amplified fragment is ligated into an vector, preferably the pCR(trademark)II vector (Invitrogen) and the sequence is determined by cycle-sequencing. Complete isolation of the coding region and the flanking sequences of the DNA polymerase gene can be performed by restriction fragmentation of the Anaerocellum thermophilum DNA with another restriction enzyme as in the first round of screening and by inverse PCR (Innis et al., (1990) PCR Protocols; Academic Press, Inc., p. 219-227). This can be accomplished with synthesized oligonucleotide primers binding at the outer DNA sequences of the gene part but in opposite orientation. These oligonucleotides, described by SEQ ID Nos. 3 and 4, were designed on the basis of the sequences which were determined by the first above described PCR. As template Anaerocellum thermophilum DNA is used which is cleaved by restriction digestion and circularized by contacting with T4 DNA ligase. To isolate the coding region of the whole polymerase gene, another PCR is performed using primers as shown in SEQ ID Nos. 5 and 6 to amplify the complete DNA polymerase gene directly from genomic DNA and introducing ends compatible with the linearized expression vector.
SEQ ID NO. 1:
Primer 1: 5xe2x80x2-WSN GAY AAY ATH CCN GGN GT-3xe2x80x2
SEQ ID NO. 2:
Primer 2: 5xe2x80x2-NCC NAC YTC NAC YTC NAR NGG-3xe2x80x2
SEQ ID NO. 3:
Primer 3: 5xe2x80x2-CAA TTC AGG GCA GTG CTG CTG ATA TC-3xe2x80x2
SEQ ID NO. 4:
Primer 4: 5xe2x80x2-GAG CTT CTG GGC ACT CTT TTC GCC-3xe2x80x2
SEQ ID NO. 5:
Primer 5: 5xe2x80x2-CGA ATT CGG CCG TCA TGA AAC TGG TTA TAT TCG ATG GAA ACA G-3xe2x80x2
SEQ ID NO. 6:
Primer 6: 5xe2x80x2-CGA ATT GGA TCC GTT TTG TCT CAT ACC AGT TCA GTC CTT C-3xe2x80x2
The gene is operably linked to appropriate control sequences for expression in either prokaryotic or eucaryotic host/vector systems. The vector preferably encodes all functions required for transformation and maintenance in a suitable host, and may encode selectable markers and/or control sequences for polymerase expression. Active recombinant thermostable polymerase can be produced by transformed host cultures either continuously or after induction of expression. Active thermostable polymerase can be recovered either from host cells or from the culture media if the protein is secreted through the cell membrane.
It is also preferable that Anaerocellum thermophilum thermostable polymerase expression is tightly controlled in E.coli during cloning and expression. Vectors useful in practicing the present invention should provide varying degrees of controlled expression of Anaerocellum thermophilum polymerase by providing some or all of the following control features: (1) promoters or sites of initiation of transcription, either directly adjacent to the start of the polymerase gene or as fusion proteins, (2) operators which could be used to turn gene expression on or off, (3) ribosome binding sites for improved translation, and (4) transcription or translation termination sites for improved stability. Appropriate vectors used in cloning and expression of Anaerocellum thermophilum polymerase include, for example, phage and plasmids. Example of phage include lambda gt11 (Promega), lambda Dash (Stratagene) lambda ZapII (Stratagene). Examples of plasmids include pBR322, pBTac2 (Boehringer Mannheim), pBluescript (Stratagene), pET3A (Rosenberg, A. H. et al., (1987) Gene 56:125-135), pASK75 (Biometra) and pET11C (Studier, F. W. et al. (1990) Methods in Enzymology, 185:60-89). According to the present invention the use of a plasmid has shown to be advantageously, particularly pASK75 (Biometra). The Plasmid pASK75 carrying the Anaerocellum thermophilum DNA polymerase gene is then designated pAR10.
Standard protocols exist for transformation, phage infection and cell culture (Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press). Of the numerous E. coli strains which can be used for plasmid transformation, the preferred strains include JM110 (ATCC 47013), LE392 pUBS 520 (Maniatis et al. supra; Brinkmann et al., (1989) Gene 85:109-114;), JM101 (ATCC No. 33876), XL1 (Stratagene), and RR1 (ATCC no. 31343), and BL21 (DE3) plysS (Studier, F. W. et al., (1990) Methods in Enzymology, supra). According to the present invention the use of the E. coli strain LE392 pUBS 520 has shown to be advantageously. The E. coli strain7221 LE392 pUBS 520 transformed with the plasmid pASK75 carrying the Anaerocellum thermophilum DNA polymerase gene (designated pAR10) is then designated E.coli AR220 (DSM No. 11177). E.coli strain XL1. Blue (Stratagene) is among the strains that can be used for lambda phage, and Y1089 can be used for lambda gt11 lysogeny. The transformed cells are preferably grown at 37xc2x0 C. and expression of the cloned gene is induced with anhydrotetracycline.
Isolation of the recombinant DNA polymerase can be performed by standard techniques. Separation and purification of the DNA polymerase from the E.coli extract can be performed by standard methods. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific interaction such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography and methods utilizing a difference in isoelectric point such as isoelectric focussing electrophoresis.
The thermostable enzyme of this invention may be used for any purpose in which such enzyme activity is necessary or desired. In a particularly preferred embodiment, the enzyme catalyzes the nucleic acid amplification reaction known as PCR. This process for amplifying nucleic acid sequences is disclosed and claimed in EP 0 201 189. The PCR nucleic acid amplification method involves amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids and produces double stranded DNA. Any nucleic acid sequence, in purified or nonpurified form, can be utilized as the starting nucleic acid(s), provided it contains or is suspected to contain the specific nucleic acid sequence desired. The nucleic acid to be amplified can be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, from natural DNA or RNA from any source, including bacteria, yeast, viruses, organelles, and higher organisms such as plants and animals, or from preparations of nucleic acids made in vitro. DNA or RNA may be extracted from blood, tissue material such as chorionic villi, or amniotic cells by a variety of techniques. See, e.g., Maniatis T. et al., 1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp. 280-281. Thus the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single-stranded or double-stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized.
The amplification of target sequences in DNA or from RNA may be performed to proof the presence of a particular sequence in the sample of nucleic acid to be analyzed or to clone a specific gene. DNA polymerase from Anaerocellum thermophilum is very useful for these processes. Due to the fact that the DNA polymerase from Anaerocellum thermophilum requires Mg++ ions as a cofactor instead of Mn++ like the other DNA polymerases from thermophilic organisms with reverse transcriptase activity of the state of the art the RNA templates can be copied with higher fidelity. These properties make DNA polymerase from Anaerocellum thermophilum a very useful tool for the molecular biologist. DNA polymerase from Anaerocellum thermophilum may also be used to simplify and improve methods for detection of RNA target molecules in a sample. In these methods DNA polymerase from Anaerocellum thermophilum catalyzes: (a) reverse transcription, (b) second strand cDNA synthesis, and, if desired, (c) amplification by PCR. The use of DNA polymerase from Anaerocellum thermophilum in the described methods eliminates the previous requirement of two sets of incubation conditions which were necessary due to the use of different enzymes for each step. The use of DNA polymerase from Anaerocellum thermophilum provides RNA reverse transcription and amplification of the resulting complementary DNA with enhanced specificity and with fewer steps than previous RNA cloning and diagnostic methods.